There have emerged powerful tools, e.g., Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination (HR) with investigator-provided donor DNA or induce gene mutations at the site of cleavage in absence of a donor by non-homologous end joining (NHEJ), both in plant and mammalian cells, including human cells. ZFNs or TALENs are formed by fusing zinc finger proteins (ZFPs) or transcription activator-like effectors (TALEs) to the non-specific cleavage domain of FokI restriction enzyme. ZFN-mediated (or TALEN-mediated) gene targeting yields high gene modification efficiencies (>10%), in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs (or TALENs). Mechanism of DSB by ZFNs (or TALENs) requires that two ZFN (or TALEN) monomers bind to their adjacent cognate sites on DNA and the FokI nuclease domains dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs (or TALENs) fused to wild-type FokI cleavage domains, homodimers may also form, which could limit the efficacy and safety of the ZFNs (or TALENs) by inducing off-target cleavage. Obligate heterodimer variants of FokI cleavage domain for creating custom ZFNs (or TALENs) are known.
However, there is a need for more efficacy and efficiency of the re-engineered obligate heterodimer variants of FokI cleavage domain with minimal cellular toxicity.